A current paradigm in biology is that so-called basal cells, present in pseudostratified epithelia, are never in contact with the luminal side of an organ. In contrast to this dogma, we recently showed that these cells extend slender body projections that cross the tight-junction barrier to reach the lumen (Shum et al. Cell 135;1108-1117, Dec 2008). This research proposal is driven by this paradigm-shifting discovery. We found that basal cells scan the luminal side of selected epithelia and modulate their function via crosstalk with other epithelial cells. We observed "apical-reaching" basal cells in several tissues of the male reproductive and upper respiratory tracts indicating that the luminal sampling property of basal cells is a generalized phenomenon. In this research program, we will examine the spatial, temporal and motional behavior of basal cells in vivo and we will characterize the intercellular communication networks between basal cells and adjacent cells. To do so, we will generate novel mice expressing the red fluorescent protein, mRaspberry, in basal cells exclusively. We will cross-breed these mice with current mice available in our laboratory, including CD11c-YFP mice that express the yellow fluorescent protein in dendritic cells, and B1-EGFP mice that express the green fluorescent protein EGFP in epididymal clear cells, non- ciliated cells of the lung, and kidney intercalated cells. This will generate a novel animal model in which several cell types will be imaged simultaneously, in live animals, using intravital multiphoton microscopes equipped with miniaturized objectives, and in which ionic and nitric oxide fluxes will be measured in real time using selective microelectrodes. We will focus on two epithelia, the epididymis, which is at the core of our research program, and the trachea. The epididymis, which connects the testis to the vas deferens, is involved in the maturation and storage of spermatozoa and, therefore, plays a crucial role in male fertility. The other target tissue of this application, the trachea, is constantly invaded by foreign allergenic and pathogenic substances, and provides a structural barrier between the airway and the body. We propose that basal cells are front-line sensors that probe luminal factors that regulate male fertility in the epididymis, and inhaled molecules in the trachea. A better understanding of the novel apical sensory role of basal cells and how they transmit their findings to adjacent cells will help define the pathophysiological mechanisms underlying male infertility, and diseases of the lung, including asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Monitoring and decoding intercellular conversations in the epididymis and trachea will, thus, promote innovative diagnostic and therapeutic interventions for the treatment of these diseases. In addition, this research program will have broader implications for our understanding of epithelia in general, as data generated here will provide unprecedented insights into the communication network established by complex tissues and on how it is perturbed in disease. PUBLIC HEALTH RELEVANCE: Many organs in the body, including those of the reproductive tract and the lungs, are comprised of a system of tubules lined by cells that form an epithelium, a structure that creates a barrier between the blood side of the organ and the cavity formed by the tube. The prevailing view is that so-called "basal cells" in these epithelia are never in contact with the fluid or air-filled cavity (known as the lumen), but we showed recently that these cells in fact extend long, slender projections that scan the lumen and modulate organ function by communicating their findings to adjacent cells. We propose to create new model systems in which the three-dimensional relationship and functions of different epithelial cell types (identified in live animals by the presence of different colored fluorescent markers) can be monitored in real time as the basal cells detect and respond to various drugs, hormones, chemicals and pathogens that appear in the cavity of the organ;the data we generate will suggest new diagnostic and therapeutic strategies for diseases including male infertility, chronic obstructive airway disease and cystic fibrosis.